Abstract: The differences in mechanical properties of normal and diseased tissues have been studied as possible contributors to various disease processes. In the case of liver fibrosis, the stiffening plays a causal role in the cell and matrix changes that lead to fibrotic disease because liver stiffness increases before cells alter their phenotype, and pharmacologically reversing stiffening inhibits the cellular processes driving fibrosis. Severe fibrosis (cirrhosis) is a precondition for the development of hepatocellular carcinoma (HCC) in the majority of chronic liver disease patients in the U.S., and the stiffness of the liver is highly correlated with the risk of HCC. Therefore, this project extends our previous studies of liver mechanics to quantify viscoelasticity and porosity of normal, premalignant (cirrhotic), and malignant liver tissue measured at the magnitudes of deformation and compressive stresses characteristic of the diseased states. Results from this study will (1) clarify the basis for the frequent clinical observations of pre-malignant stiffening, and (2) mechanically define microenvironments within cirrhotic livers. Fresh human samples and well-defined mouse models will be obtained from Core 1, and rheologic data will be used to test theoretical models of Core 2 to develop a molecular and structural understanding of the determinants of liver stiffening in cirrhosis. Results generated from goal (1) within the first 1-2 years of the grant period will be of immediate clinical interest to diagnostic interpretations of emerging clinical measurements of pre-malignant liver stiffening. Goal (2) is key to formulating culture systems and cell-level questions that constitute one aim of Project-1 and a larger part of Projects-2 & 3. These studies using purified 2D and 3D culture systems with precisely tuned physical and chemical properties will reveal the cellular and molecular mechanisms triggered by the stiffened, cirrhotic state that create an environment conducive to tumor growth. Integration of results from human tissues with those that are recapitulated in mouse models is necessary for controlled culture systems in which it is possible to molecularly dissect the likely roles of physical confinement, pressures, and tissue defects.